Lividomycin b derivatives

ABSTRACT

DERIVATIVES OF LIVIDOMYCIN B HAVE BEEN PREPARED WHICH POSSESS SUBSTANTIALLY IMPROVED ANTIBACTERIAL ACTIVITY. AN EXAMPLE OF SUCH AN AGENT IS 1-(L-( - )-Y-AMINO-A-HYDROXYBUTYL)-LIVIDOMYCIN B (IV, BB-K82).

Patented Apr. 30, 1974 3,808,198 I LIVIDOMYCIN B DERIVATIVES Takayuki Naito and Susumu Nakagawa, Tokyo, and

Soichiro Toda, Kasukabe, Japan, assignors to Bristol- Myers Company, New York, NY. No Drawing. Filed Sept. 7, 1972, Ser. No, 287,176

Int. Cl. C07c 129/18 US. Cl. 260-210 AB 6 Claims ABSTRACT OF THE DISCLOSURE Derivatives of lividomycin B have been prepared which possess substantially improved antibacterial activity. An example of such an agent is 1-[L-( -'y-amino--hydroxybutyryl]-lividomycin B [IV, BB-K82].

BACKGROUND OF THE INVENTION 1) Field of the invention-This invention relates to a semisynthetic l-substituted derivative of lividomycin B, said compound being prepared by acylating the l-aminofunction of lividomycin B with a -amino-a-hydroxybutyryl moiety.

(2) Description of the prior art-(A) The lividomycins are reported and described in the Journal of Antibiotics (Japan) 24, No. 6, pp. 333-346 (1961). -In particular lividomycin A and B are reported as fermented from Streptomyces lividus nov. sp., a culture deposited in the American Type Culture Collection at Rockville, Md., as A.T.C.C. No. 21178 and in the Fermentation Research Institute, Agency of Industrial Science 8: Technology, Chiba, Japan, as FERM-P No. 50.

(B) Lividomycin B has been prepared from Lividomycin A by T. Mori et al., J. Antibiotics, 25, 149 (1972).

SUMMARY OF THE INVENTION The compound having the formula in which R is L-( )-'y-amino--hydroxybutyryl; or a nontoxic pharrnaceutically acceptable acid addition salt thereof is a valuable antibacterial agent.

This invention relates to a semi-synthetic derivative of lividomycin B, said compound being known as 1-[L-()- 'y-amino-a-hydroxybutyryl]-lividomycin 'B and having the formula in which R is L-( )-'y-amino--hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

For the purpose of this disclosure, the term nontoxic pharmaceutically acceptable acid addition salt" shall mean a mono, di, tri, tetra or penta salt formed by the interaction of one molecule of Compound IV with 1-5 moles of a nontoxic, pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine containing pharmaceuticals.

' Lividomycin B is a compound having the formula by the following diagrammatic scheme:

N-(benzoyl oxycarbonyloxy Succinimide (1) Lividomycin B y no 2 HO H (2) Compound II N hydroxysuccinlmide ester of L- )-'y-benzyloxycarbonylaminoa-hydroxybutyric acid A preferred embodiment of the present invention is the compound having the formula no C3 01 BO H in which R is H or and R is H or iL-(-)-'y-amino-a-hydroxybutyryl or L-()-'y-benzyloxycarbonylamino a hydroxybutyryl wherein R or R must be other than H; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

Another preferred embodiment is the compound of Formula V wherein R is H and R is L-( )-'y-amino-ahydroxybutyryl; or a nontoxic pharmaceutically acceptable =acid addition salt thereof.

Other most preferred embodiments are the sulfate, hydrochloride, acetate, maleate, citrate, ascorbate, nitrate or phosphate salt of Compound V.

Another more preferred embodiment is the monosulfate salt of Compound IV.

Still another most preferred embodiment is the disulfate salt of Compound IV.

The objectives .of the present invention have been achieved, by the provision according to the present invention of the process for the preparation of the compound having the formula in which R is L-()-'y-amino-a-hydroxybutyryl; or a non-toxic pharmaceutically acceptable acid addition salt thereof; which process comprises the consecutive steps of (A) treating lividomycin B with an agent selected from the compounds having the formulas o R O wain 1;-

om-(tax Rs CH3 0 cm-Jf-o-ii-m 02NX CH: 1

o x-cm-iLon (or a carbodiimide addition compound thereof) or (or a carbodiimide addition compound thereof), in which R and R are alike or different and each is H, F, Cl, Br, N0 OH, (lower)alkyl or (lower-)alkoxy, X is chloro, bromo or iodo, or a functional equivalent as a reactant; in a ratio of one mole or less of agent per mole of lividomycin B in a solvent, preferably selected from the group comprised of dimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, methanol, ethanol, water, acetone, pyridine, N-(lower)alkylpiperidine, or mixtures thereof, but preferably 1:1 watertetrahydrofuran, at a temperature below 50 C. and preferably below 25 C., to produce the compound having the formula in which R and R are as defined above;

(B) acylating Compound 11 with an acylating agent having the formula OH O WNH-CHzCHf-('JH-i-M VII in which W is a radical selected from the group comprising 0 d-om-cm-rixoH,( or

but preferably @cmob R6 M is a radical selected from the group comprising in which R and R are as above; in a ratio of at least 0.5 mole of Compound V11 per mole of Compound II, but preferably in a ratio of about 0.5 to about 1.4, and most preferably in a ratio of about 0.8 to about 1.1, in a solvent preferably selected from the group comprising a mixture of water and ethyleneglycol dimethyl ether, dioxane, di-

6 methylacetamide, dimethylformamide, tetrahydrofuran, propyleneglycoldimethyl ether, or the like but preferably 1:1 water-tetrahydrofuran, to produce the compound of the formula @wm-o-E- by hydrogenating Compound HI with hydrogen in the presence of a metal catalyst, preferably selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, but preferably palladium, and most preferably palladium on charcoal, in a waterwater miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether, propyleneglycol dimethyl ether, or the like, but preferably 1:1 water-dioxane.

It should be apparent to those knowledgeable in the art that other agents can be used in the process above to acylate the amine functions of the intermediate compounds of the instant invention. This disclosure is meant to include all such acylating agents that produce labile amine blocking groups, said labile blocking groups commonly employed in the synthesis of peptides. The labile blocking groups must be readily removable by methods commonly known in the art. Examples of said labile blocking groups and their removal can be found in the review of A. Kapoor, J. Pharm. Sciences 59, pp. 1-27 (1970'). FUI1 tional equivalents acylating agent for primary amine groups would include corresponding carboxylic chlorides, bromides, acid anhydrides, including mixed anhydrides and particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carbonic acid, of alkyland aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active ester of thioester (e.g., with p-nitrophenol, 2,4dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with Compound 11 after first reacting said free acid with N,N dimethylchloroforminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI/ 6, 360 1965 or by the use of enzymes or of an N,N'-carbonyldiimidazole or and N,N'-carbonylditriazole [cf. Sheehan and Hess, J. Amer. Chem. Soc. 77, (1955)] or of alkynylamine reagent [cf. R. Buijile and H. G. Viehe, Angew., Chem., International Edition 3, 582 (1964)], or of a ketenimine reagent [cf. C. 1.. Stevens and M. Monk, J. Amer. Chem. Soc., 80, 4065 (1958)] or of an isoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofs'on-and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)]. Another equivalent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid which amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room tempera ture in tetrahydrofuran, chloroform, dimcthylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield diimidazolides. The by-product, imidazole, precipitates and may be separated and the imidazolide isolated, but this is not essential. These reactions are wellknown in the art [cf. US. Pat. Nos. 3,079,314, 3,117,126 and 3,129,224 and British Pat. Nos. 932,644, 957,570 and 959,054).

Compound :IV, 1 [L-()-' -amino-u-hydroxybutyryl]- lividomycin B, possesses excellent antibacterial activity. Illustrated below is a table showing the minimal inhibitory concentrations (MICs) of lividomycin B, Compound IV 5 them are lividomycin B derivatives acylated with I.-

HABA. Although the position of L-HABA residue on each of the products has not been established yet, BBKSO to -BB-K83 are assumed to the monoacyl derivatives of lividomycin B and BBK84 to BBK86 diacyl derivatives 10 'based on their behaviors in thin layer and column chromatographies.

BBB82 is the most active component among the acylated products of lividomycin B and, in addition, exhibits a considerably increased activity compared with that of the parent antibiotic against the four =kanamycin-desistant organisms as described above. On the basis of the data we have accumulated on BBK82 and as compared to the properties of L-HABA derivatives of other aminoglyco- (BB-K82) and other related mono and diacylated deriva- 5 tives of lividomycin B against a variety of gram-positive and gram-negative bacteria as determined by the two-fold agar dilution method.

IN VITRO ANTIMICROBIAL ACTIVITIES F L-EABA DERIVATIVES OF LIVIDOMYCIN B BIB-K81 Livido MIC (meg/ml.) BB-KSO BBK81 (lot 1-2) BBK83 BBK84 BIB-K85 BB-K86 mycin B Ee-l E. coli NIHJ' 6.3 100 0.8 6.3 100 6. 3 100 1. 6 Ec-3 E. coli Juhl A15119 12.5 100 0. 8 6.3 100 12.5 100 31 130-4 E. coli 1115169.-.- 12.5 100 0. 8 6.3 100 12. 5 100 3. 1 Ec5 E. coli KM-R 1 A2036 100 100 0. 8 6. 3 100 12. 5 100 100 Ec-6 E. coli A98 3. 1 100 0. 4 6. 3 100 6. 3 100 1. 6 Ec-7 E. coli KM-R 1 A20365 25 50 0. 2 1. 6 100 1. 6 100 100 EO-B E. coli K-12 A9632 6. 3 100 0. 8 6. 3 100 6. 3 100 1. 6 130-9 E. coli K-12 KM--R 1120664..-- 6.3 100 0.8 3. 1 100 6. 3 100 1. 6 Ec-10 E. coli K42 KM-R 1 1120665-..- 50 100 0.4 3. 1 100 3. 1 100 100 Ec-52 E. coli W677 A20684 12. 5 100 0. 4 3.1 100 6. 3 100 1. 6 Ec-53 E. coli J R/W677 A20683 6. 3 100 0. 8 6. 3 100 6. 3 100 3, 1 Kp-l K. D-11- 0. 8 0. 1 0. 8 12. 5 0.4 100 0.2 Kp-8 K. pneumoniae Type 22 1120680 12.5 100 1. 6 6. 3 100 12.5 100 1. 6 8111-1 Ser. Mar A200l9 125 100 0.8 6. 3 100 6. 3 100 1.6 Pa-1 P3. aer D 100 100 6. 3 100 50 100 25 Pa-4 Pa. aeruainosa H9 D-113 KM-R 1 100 100 25 100 100 100 100 25 Pa-2 P3. aemqinoaa A9923 100 100 25 100 100 100 100 25 Pit-3 Pa. aeruginosa A9930 3. 1 25 0. 2 3.1 100 3. 1 100 0, 8 P8-5 P3. aer' A15150... 100 100 25 100 100 100 100 50 Pa-6 P8. Gm A15104.-- 100 100 12.5 100 100 100 100 25 Pa-15 Pa. aeruginosa GM-R 1 A20717 100 100 12. 5 100 100 100 100 50 Pa-16 P8. aeruginosa GM-ZR 1120718-..- 100 100 25 100 100 100 100 100 Pa-17 P8. aemgi'lwsu H6 D-114 NM-R.-- 50 100 6. 3 100 100 100 100 6.3 Pv-l Pr. vuluarz's A9436- 1. 6 50 0. 8 1. 6 100 3. 1 100 0. 4 Pv-2 Pr. vulaaris A9526. 6. 3 100 0. 8 6. 3 100 6. 3 100 1. 6 Pin-1 Pr mirabilis A9554 6. 3 100 1. 6 12. 5 100 12. 5 100 1. 6 Pin-2 Pr mirabilts A9900 6.3 100 1. 6 6.3 100 6.3 100 1.6 Pg-l Pr. mtrgam'i A9553- 3. 1 100 0. 8 3. 1 100 3. 1 100 0. 8 Pg-2 Pr. mirganii A20031 6. 3 100 1. 6 6. 3 100 6. 3 100 1. 6 Pr-l Pr. rettqeri A15167 Sa-2 S. aureus Smith A15167 1. 6 12. 5 0.1 1. 6 25 1. 6 100 0. 2 Sa-4 S. aureus 209P SM-R 12.5 100 0. 8 12- 5 100 12.5 100 1. 6 Sa-lO S. aureus KM-R 5 A20239 25 50 0- 2 3. 1 100 1. 6 100 100 Rs-l B. aubtilz's PCI219....- M6-1 Mycob. 607 1. 6 6. 3 0.2 1. 6 12. 5 0. 4 12. 5 0. 4 M6-2 Mycob. 607 KMR 50 100 6. 3 50 100 6. 3 100 6. 3 M6-3 Mycob. 607 KM-RL 25 100 6.3 12.5 100 3.1 100 3.1 Mp-l Mycob. phlci 0- 8 1. 6 0. 1 1. 6 1. 6 0. 2 12. 5 0. 1 Mr-l Mycob. ranae 3. 1 6. 3 0.2 3. 1 12. 5 0. 4 25 0. 4

1 KM=Kanamycin resistant. i GM= Gentamycin resistant. 3 SM=Streptomycin resistant.

BB-K82, the most active component, showed much side antibiotics prepared in our laboratories, it has been better activity (125-500 times) than the parent antibiotic, lividomycin B, against four of kanamycin-resistant organism (E. coli A20363, E. coli A20365, E. coli A20665 and determined the acylation site of BB-K82 is the l-amino group of the 2-deoxystreptamine moiety of lividomycin B, 1.e.

S. aureus A20239) which are known to inactivate kanamycin by 3'-phosphorylation (and also lividomycin probably by phosphorylation at a different position). BBK82 also indicated the in vitro activity about equal to that of lividomycin B against other test organisms including two gentamicin-resistant strains (E. coli A20683 and K. pneumoniae A2068'0) which are known to inactivate gentami- 5 cin C by 2-adenylation.

BBKSO, BBK83 and BBKSS showed an antibacterial spectrum similar to that of BB-K82 against the bacteria tested so far including the four lividomycinresistant and BB-K82-sensitive strains, although the MIC values are 4-16 times inferior to those of BB-K82.

BB-KSI, BBK84 and BBK86 were almost inactive against most of the test organisms.

It has been confirmed by thin layer chromatography (TLC) that all of the products, BBK80 through BBK86,

Compound -1V is valuable as an antibacterial agent, nutritional supplement in animal feeds, therapeutic agent 9 in poultry and animals, including "man; and is especially valuable in the treatment of infectious diseases causedby Gram-positive and Gram-negative bacteriaq I 7 Compound IV when administered orally is useful as an adjunctive treatment for preoperative sterilization ofthe bowel. Both aerobic and anaerobic flora which are susceptible to this drug are reduced in the large intestine. When accompanied by adequate mechanical cleansing, it is useful in preparing for colonic surgery. I v

Compound IV is elTective in the treatment of 'ysternic bacterial infections when administered parenterally in the dosage range of about 250 mg. to about 3000 mg. per day in divided doses three or four times a day. Generally the compound is effective when administered at a dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

Preparation of Lividomycin B Lividomycin B was prepared from lividomycin A by a slightly modified procedure of the chemical conversion reported by T. Mori et al., J. Antibiotics, 25, 149 (1972), which comprised N-acetylation of lividomycin'A followed by degradation of the mannose moiety with periodate and subsequent hydrolysis with barium hydroxide according to the scheme below: 7 i

lividomycin A (time);

Lividomycin B A suspension of 5.99 g. of lividomycinA in 300 ml.

of methanol was stirred with 30 ml. of acetic anhydride overnight at room temperature. After confirming the negative ninhydrin test, the solution was evaporated into dryness. The oily residue was triturated with ether 'to give a white powder of penta-N-acetyl lividomycin A in an almost quantitative yield.

A solution of the penta-acetate in water (750 ml.) was treated with sodium periodate (4.24 g.) with stirring in the dark at room temperature for 18 hours. The reaction mixture was treated with ethylene glycol (2 ml.) for two hours at room temperature and freed fromiodate by precipitation with lead diacetate (4.0 g.), the excess lead ions being removed with 5% sulfuric acid (2 ml.). The filtrate was heated with 10% AcOH (20 ml.) and phenylhydrazine (20 ml.) on a boiling water bath for three hours. After the reaction mixture was extracted with chloroform,

lite IR-120, (H+, 110 ml.) and Amberlite IRA-410 (OI-I, 160 ml.). The eluate was concentrated to dryness to give yellow powder (6.57 g). A mixture of the crude powder (6.5 g.) and barium hydroxide octahydrate (175 g.) was refluxed with stirring for five hours. The reaction mixture was cooled and filtered to remove insoluble material. To the filtrate was added ammonium carbonate (48 g.) and the mixture filtered to remove the resultant barium carbonate. The filtrate was concentrated to dryness to give 3.8 g. of crude lividomycin B. The crude material was purified by column chromatography with Amberlite CG-50 (NH.,**, 65 ml.) to give 2.75 g. of pure lividomycin B (60% yield from penta-N-acetyl lividomycin A).

Amberlite CG 50 is the trade name for the chromatographic grade of a weakly acidic cationic exchange resin of a carboxylic-polymethacrylic type.

Amberlite IRA-410 is the tradename for a strongly basic anion exchange resin which is an amination produce between dimethylethanolamine and a ehloromethylated styrene-divinylbenzene copolymer and its contains 3-5% of bound divinylbenzene. Mesh size 20-50. U.S. Pat. Nos. 2,614,099 and 2,591,573.

Amberlite IR-l20 is the tradename for a high density nuclear sulfonic acid type cationic exchange resin supplied in either hydrogen or sodium form as beadsl6-50 mesh.

EXAMPLES Lividomycin B was acylated with 1 ()-'y-amino-ahydroxybutyric acid using the scheme illustrated below to produce seven derivatives (four monoacylated and three diacylated) which were designated BB-KSO through BB- K86 in the order of elution in column chromatography with CG-5O ion exchanger. BB-K82, which exhibited the best antibacterial activity, is the desired l-N-acyl derivative.

Lividomycin B 6"-mono-Cbz-Llvidomycin B O I CbZ-NHCHrCHzCH-COO-N l H monoacyl derivatives dlacyl derivatives B B EXAMPLE 1 I Preparation of L- -'y-benzyloxycarbonylamino-ahydroxybutyric acid (VI) L (-)-'y-amino-u-hydroxybutyric acid (7.4 g., 0.062

mole) was added to a solution of 5.2 g. (0.13 mole) of sodium hyroxide in 50 ml. of water. To the stirred soluthe aqueous layer was passed through columns of Am'bertion was added dropwise at 0-5 C. over a period of 0.5 hour, 11.7 g. (0.068 mole) of carbobenzoxy chloride and the mixture was stirred for one hour at the same temperature. The reaction mixture was washed with 50 ml. of ether, adjusted to pH 2 with dilute hydrochloric acid and extracted with four -ml. portions of ether. The ethereal extracts were combined, washed with a small amount of saturated sodium chloride solution, dried with anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo and the resulting residue was crystallized from benzene to give 11.6 g. (74%) of colorless plates; melting point 78.5-79.5 0., [011 -4.5 (c.=2, CH OH). Infrared (IR) [KBr]: 'y 1740, 1690 MIL-1. Nuclear magnetic resonance (NMR) (acetone-d 6 (in p.p.m. from TMS) 2.0 (2H, m), 3.29 (2H, d-d, J= 6.7 and 12 Hz.), 4.16 1H, d-d, 1:45 and 8 Hz), 4.99 (2H, s), 6.2 (2H, broad), 7.21 (5H, s).

Analysis.-Calcd for C H NO (percent): C, 56.91; H, 5.97; N, 5.33. Found (percent): C, 56.66; H, 5.97; N,

EXAMPLE 2- N-hydroxysuccinimide ester of L-()-'y-benzyloxycarbonylamino-a-hydroxybutyric acid (VII) A solution of 10.6 g. (0.042 mole) of VI and 4.8 g. (0.042 mole) of N-hydroxysuccinirnide in 200 ml. of ethyl acetate was cooled to C. and then 8.6 g. (0.042 mole) of N,N-dicyclohexylcarbodiimide was added. The mixture was kept overnight in a refrigerator. The dicyclohexylurea which separated was filtered ofi? and the filtrate was concentrated to about 50 ml. under reduced pressure to give colorless crystals of VII which were collected by filtration; 6.4 g./M.P. 121122.5 C. The filtrate was evaporated to dryness in vacuo and the crystalline residue waswashed with 20" ml. of a benzene-n-hexane mixture to give an additional amount of VII. The total yield was 1.34 g. 92% [[1] 1.5 (6.:2, CHCl IR (KBr) 7 1810, 1755, 1740, 1680 cmr NMR (acetone-d (in p.p.m. from TMS) 2.0 (2H, m), 2.83 (4H, s), 3.37 (2H, d-d, J=6.5 and 12.5 Hz.), 4.56 (1H, m), 4.99 (2H, s), 6.3 (2H, broad), 7.23 (H, s). p

Analysis.Calcd for C H N O percent): C, 54.85; H, 5.18; N, 8.00. Found (percent): C, 54.79, 54.70; N, 5.21, 5.20; N, 8.14, 8.12.

Example 3.-Preparation of 6"'-benzyloxycarbonyl lividomycin B (H) To a stirred solution of 2.75 g. (4.57 mmole) of lividomycin B free base in 65 m1. of 45%' aqueous THF (tetrahydrofuran) was added 1.14 g. (4.57 mmole) of N-benzyloxycarbonyloxysuccinimide at C. The reaction mixture was stirred for five hours at room temperature and evaporated under reduced pressure to remove the organic solvent. The resultant aqueous solution was washed with n-butanol and evaporated to dryness under reduced pressure to give 3.0 g. of crude solid. The crude material was dissolved in a small amount of water and charged on a column of CG-SO (NH 100 ml.). The column was washed with 150 ml. of water and eluted with 0.1 NH OH. The eluate was collected in 20-ml. frao-' tion. Fractions 13 to 29 were combined, evaporated under reduced pressure and lyophilized to give 1.023 g. (30.5%) of 6"'- benzyloxycarbonyl lividomycin B which did not contain dividomycin B itself. w 1700 crn.- The prod uct was usedfor the next reaction without further purifi-' cation.

Example 4.-Preparation of 1- [L-()-'y-amino-'a-hydroxybutyryl]-lividomycin B (IV, BB-K82) and BB-KSO, 81, 83-86 ladium on charcoal at room temperature and atmospheric 1 G. W. Anderson et al., J. Am. Chem. 800., 8641839 (1964). 8

pressure. The reaction mixture was filtered and-evaporated under reduced pressure to remove the organic.sol-

vent. The resultant aqueous solution was adsorbedona column of CG-50 (NI-I 70 ml.)..The column was, washed with 120 ml. of water and irrigated successively with 1.1 l. of 0.1 N ammonia, 1.5 l. of 0.2 N ammonia,-

ninhydrin). The fractions belonging to the same cut werecom'bined, concentrated under reduced pressure and lyophilized.

Wt. Fraction isolated, Cut number Eluted withmg. Compound 0 1 N NHiOH 82 BB-KSO. 0 2 N NHlOH 460 Lividornycin B. 0 3 N NHAOH 77 BB-K81. 0 3 N NHrOH 68 BB-KSZ 0 5 N NHaOH 99 BB-K88 1 ON NH4OH 84 IKE-K84 1.0 N NHAOH 57 BB-K85 1.0N NILOH 21 BB-K86 *Rechromatography with CG-50 (NH4-*, 7 ml.) gave 45 mg. of a pure product designated BB-K82, lot 1-2.

PROPERTIES M.P. C. (KBI), (H20) Code number (den) R1 1 cm. degrees BIB-K86.-. 184-189 3 0. 09 1, 650

1 BB-K83 is active against some lividomyein-resistant organisms, while BB-KSI is not.

BB-K85 is active against some lividomycin-resistant organisms, while BIZ-K8615 not.

Microanalysis of BB-K82, lot 1-2 AnalysiS.-Calcd for C27H52N015-3H2C03.2H2O (percent): C, 39.04; H, 6.77; N, 9.11. Found (percent): C, 39.16; H, 6.55; N, 9.12.

Example 5.-Preparation of N-(benzyloxycarbonyloxy) succinimide N-hydroxysuccinimide (23 g., 0.2 mole) was dissolved in a solution of 9 g. (0.22 mole) of sodium hydroxide in 200 ml. of water. To the stirred solution was added dropwise 34 g. (0.2 mole) of carbobenzoxychloride with watercooling and then the mixture was stirredat room temperature overnight to separate the carbobenzoxy derivative which was collected by filtration, washed with water and air dried. Yield 41.1 g. (82%). Recrystallization from benzene-n-hexane-(lOzl) gave colorless prisms melting at 78+79 C.

Example 6. Preparation of L-()-'y-amino-u-hydroxybutyric acid from ambutyrosin A or B ;or mixtures thereof v 13 drying. The L-(-)-'y-amino-u-hydroxybutyric acid is characterized as a crystalline material having a MP. of 212.5-214.5 C. [column 2, lines 31-38, U.S. Pat. No. 3,541,078].

Example 7.Preparation of L-( -)--y-amino-a-hydroxybutyric acid from DL-a-hydroxy-'y-phthalimidobutyric acid (A) Dehydroabietylammonium L-a-hydroxy-y-phthalimidobutyrate.-To a solution of 25 g. (0.1 mole) of ahydroxy-y-phthalimidobutyric acid 2 in 200 ml. of ethanol was added a solution of 29 g. (0.1 mole) of dehydroabietylamine in 130 ml. of ethanol. The solution was shaken vigorously for a minute and stood at room temperature for five hours during which time fine needles crystallized out. The crystals were collected by filtration, washed with 50 ml. of ethanol and air-dried to obtain 30.1 g. (56%) of a diastereomer of the dehydroabietylamine salt. M.P. 93-94" C. [(11 +l5 (c. 2.5, MeOH).

Recrystallization from 300 ml. of ethanol gave 23.2 g.

(43%) of the pure product. M.P. 94-95 C. +10.8

(c. 2.5, MeOH). Further recrystallization did not change the melting point and the specific rotation.

Analysis.-Calcd for C H N O H O (percent): C, 69.54; H, 8.02; N, 5.07. Found (percent): C, 69.58; H, 8.08, N, 5.07.

(B) L-( )-'y-amino or hydroxybutyric acid-To a solution of 1.5 g. (0.014 mole) of sodium carbonate in 40 ml. of water were added 5.3 g. (0.01 mole) of dehydroabietylammonium-L-a-hydroxy 'y phthalimidobutyrate and 60 ml. of ether. The mixture was shaken vigorously until all of the solid had dissolved. The ether layer was separated. The aqueous solution was washed twice with 20-ml. portions of ether and evaporated to 15 ml. under reduced pressure. To the concentrate was added ml. of concentrated hydrochloric acid and the mixture was refluxed for ten hours. After cooling, separated phthalic acid was removed by filtration. The filtrate was evaporated under reduced pressure. The residue was dissolved in 10 ml. of water and the solution was evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The residual syrup was dissolved in 10 ml. of water and filtered to remove a small amount of insoluble phthalic acid. The filtrate was adsorbed on a column of IR-120 (I-I+, 1 cm. x 35 cm.), the column was washed with 300 ml. of water and eluted with 1 N ammonium hydroxide solution. The eluate was collected in -ml. fraction. The ninhydrin positive fractions 10 to 16 were combined and evaporated under reduced pressure to give a syrup which crystallized gradually. The crystals were triturated with ethanol, filtered and dried in a vacuum desiccator to give 0.78 g. (66%) of L-(-)-'y-aminoa-hydroxybutyric acid. M.P. 206-207 C. [04 -29 (C. 2.5, H 0). The IR spectrum was identical with an authentic sample which was obtained from a-mbutyrosin.

Example 8.-Preparation of monosulfate salt of l-[L- (-)-'y-amino-a-hydroxybutyryl] -lividomycin B One mole of 1-[L-()-'y-amino-a-hydroxybutyryl)- lividomycin B is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added one mole of sulfuric acid dissolved in 500 ml. of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture until precipitation occurs. The solids are collected by filtration and are determined to be the desired monosulfate salt.

Y. Saito et al., Tetrahedron Letters, 1970, 4863.

Example 9.Preparation of the disulfate salt of l-[L- w-aminoaxehydroxybutyryl] -livido-mycin B no CH OH in which R is H or and R is H, L-( )--y-amino-a-hydroxybutyryl or L-( 'y-benzyloxycarbonylamino-a-hydroxybutyryl, wherein R or R must be other than H; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

2. The compound of claim 1 wherein R is and R is H.

3. The compound of claim 1 wherein R is O mmcHro-i land R is L-(-)-' -benzyloxycarbonylamino-a-hydroxybutyryl.

4. The compound of claim 1 wherein R is H and R is L-()-'y-amino-a-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

5. The monosulfate salt of the compound of claim 4.

6. The disulfate salt of the compound of claim 4.

References Cited UNITED STATES PATENTS 2,916,485 12/ 1959 Frohardt et al. 260-210 AB 3,277,078 10/1966 Horii et al. 260-210 AB 3,661,892 5/1972 Shomura et al. 260-210 AB 3,669,838 6/ 1972 Shier et al. 260-210 AB JOHNNIE R. BROWN, Primary Examiner U.S. Cl. X.R. 

